The developmental switch in hemoglobin synthesis, from embryonic (including epsilon globin) to fetal (gamma) to adult (beta) hemoglobins, that occurs during ontogeny is of great interest because of its fundamental biology as well as its relevance to the treatment of hemoglobinopathies. We have been studying transcriptional regulation of globin genes to clarify the molecular mechanisms of this switch. Within the cell, regulation of transcriptional activity of globin genes is determined by a variety of cis- acting regulatory DNA sequences and trans-acting proteins. One of the cis- acting elements is the silencer located in the region between -177 bp and - 392 bp 5' to the epsilon globin gene, which was characterized in this laboratory. To delineate the molecular mechanism of the epsilon globin gene silencer activity and its possible role in the developmental regulation of epsilon gene expression, further characterization of epsilon gene transcription initiation sites in cell systems where the epsilon gene is up and down regulated, such as MB-02 and the two-phase liquid culture system of Fibach, was studied. A variety of clues in the literature and in our own data suggested that the epsilon silencer could affect transcription initiation. The K562 erythroleukemia cell line constitutively expresses low levels of embryonic and fetal, but not adult hemoglobin and can serve as a control. We have now shown that in NB-02 cells levels of correctly initiated epsilon transcripts are significantly lower than in K562 cells. Correctly initiated epsilon transcripts were not detectable in normal cells prepared in the liquid culture system. We have, however, observed a smaller band in T(1) analysis using a specific probe for exon I and 5' upstream region of the epsilon globin gene. Moreover, using the exon II of the epsilon gene as probe in T(1) analysis we have detected the expected band and also a few smaller bands. Quantitating of epsilon exon I transcripts using cDNA amplification showed a small amount of correctly initiated epsilon transcripts in normal cells. Further characterization of the smaller bands will test the possible role of the epsilon gene silencer in regulation of developmental expression of epsilon gene. The data so far are compatible with our original hypothesis.